Figure 4

Validation of predicted binding sites by luciferase reporter assays. Synthetic oligonucleotides encoding 60 nucleotides that encompass predicted miRNA binding sites were cloned into luciferase reporter vectors. (a) These constructs were co-transfected into HEK293T-17-5p cells with a β-galactosidase expressing plasmid, and either a 17-5p 2'-O-Me ASO or a scrambled sequence ASO. Luciferase signals were normalized to β-galactosidase signals (as a control for transfection efficiency), and the mean and standard error relative to the scrambled ASO control are shown. Constructs that show a significant increase in luciferase expression with miR-17-5p ASO treatment (p ≤ 0.05 in a Student's t-test) are indicated in black. (b) Selected constructs were co-transfected into HEK293T (wild-type cells that express very low levels of miR-17-5p) with a β-galactosidase expressing plasmid, and either a short dsRNA precursor for miR-17-5p or a negative control dsRNA precursor. Mean and standard errors of luciferase signals normalized to β-galactosidase activity are shown, and all sites except PPARA-B show significantly less luciferase activity with miR-17-5p treatment compared to control miRNA treatment (p ≤ 0.05 in a Student's t-test).